SEARCH

SEARCH BY CITATION

Keywords:

  • HEK-293;
  • FACS;
  • monoclonal antibody;
  • stable transfection;
  • cold capture

Abstract

BACKGROUND: Human embryonic kidney-293 (HEK-293) cells are commonly used as a transient expression host but their application in stable therapeutic protein production is limited. This is presumably due to the absence of a suitable amplifiable expression system and hence limited protein output compared with other mammalian cells such as Chinese hamster ovary cells. This paper describes a rapid clonal selection method for isolating HEK293 cell lines with high specific productivity, for a non-amplifiable expression system, to achieve high-level, scalable expression of recombinant antibodies.

RESULTS: Flow cytometry utilizing cold capture of secreted protein on the cell surface was applied to isolate high expressing clones from a stable antibiotic resistant pool. The top three isolated clones showed a five- to seven-fold improvement in volumetric outputs compared with the initial resistant pool (∼20 mg L−1) under batch conditions. In fed-batch conditions using commercially available hydrolysate supplements, the final titre was further increased to 500–600 mg L−1 in shaker flasks. One clone was scaled up to 25 L bag production using a similar hydrolysate feeding regime. The antibody titre reached 655 mg L−1, and 12 g of antibody was recovered after purification, demonstrating scalability of the process. The process of clonal selection through to fed-batch production of gram quantities was completed within 4 months.

CONCLUSION: HEK-293 cells can be used as a stable host for the production of biopharmaceuticals, producing gram quantities of recombinant proteins for preclinical development. Copyright © 2011 Society of Chemical Industry